1. Field of the Invention
The present invention relates generally to immunological assay techniques for determining the presence of an analyte in fluids and particularly to an immunoassay testing method utilizing cross-reactive antibodies which has particular application in the qualitative and quantitative determination of the level of unstable analytes such as beta-lactam antibiotics in fluids with particular emphasis on penicillin detection and measurement.
2. Description of Prior Art
Immunoassay methods are extensively described in literature, for example as in Methods in Enzymology, Vol. 92, Pages 377-543, (1983) Academic Press, New York. Generally, immunoassay methods require (i) specific antibodies raised against the analyte of interest, and (ii) a signal generating molecule which is either derived from the analyte of interest or from the antibody raised specifically to the analyte of interest, or another molecule which reacts with the said antigen or antibody. The antibody, the analyte of interest and the signal generating molecule may be reacted in one-phase or two-phase systems. Thereafter, the amount of signal generating molecules that are bound directly or indirectly to the specific antibody are differentiated from the unbound signal generating molecule. The proportion of bound or free molecule gives a measure of analyte present in the sample.
Several patents have been issued covering the use of various signal generating molecules (flourescence, enzyme, radioisotope, latex agglutination etc.) various techniques of conducting the assay, and ways of immobilizing antibodies.
The prior art has generally recognized and demanded that an accurate measurement technique must utilize an antibody that is specific to the analyte being measured. Such specific antibodies are raised by injecting in a living system capable of forming antibodies,
A. highly purified analyte
B. highly purified analyte conjugated to a high molecular weight substance, or,
C. a highly purified fragment of the analyte of interest as such or as a conjugated molecule
The living system responds to this "invasion" of a foreign molecule and produces specific antibodies to the analyte of interest.
In practice, one often forms cross-reacting antibodies which not only partly react to the analyte of interest but also react with other constituents which may be commonly present in the sample to be assayed. These cross-reactive antibodies may interfere in the test procedure and prevent specific measurement of the analyte. It is because of this that the prior art has generally required the use of specific antibodies.
The prior art also describes ways of minimizing the harmful effect of cross-reactive antibodies. This could be achieve by the purification of antibodies by affinity chromatography, the use of hybridoma techniques to make specific antibodies or the use of so called "sandwich" techniques. The "sandwich" approach relies on redundancy to eliminate harmful effects of cross-reactive antibodies. Thus for example, specific antibodies are raised against the analyte in two different living systems. In the actual test, the analyte is sandwiched between these two different but specific antibodies. If the cross-reactivity was resulting from the living system used to make the antibody, such an approach will eliminate cross-reactivity.
Another common "sandwich" technique involves making two pure fragments of the analyte and making specific antibodies against each fragment. In the actual assay, the intact analyte is sandwiched between the antibodies made against its fragments. Such an approach will eliminate cross-reactivity problems which occur due to the presence of antibodies against a portion of the analyte.
Such immunological techniques utilizing specific antibodies are not feasible in many situations, including the following:
(a) When the analyte could not be obtained in a pure enough form and hence specific antibodies could not be raised against it. Many delicate biochemicals undergo changes as they are purified and fall into this category.
(b) When the analyte is a sensitive hapten which could not be conjugated without altering the analyte. Beta-lactam antibiotics for example react very easily with proteins. One often needs to form a conjugate of beta-lactam antibiotics with a protein to render it immunogenic. However, the beta-lactam ring is opened during such conjugation and antibodies so raised will only cross-react with the intact antibiotic molecule and are not specific to the native antibiotic molecule.
(c) When the analyte could be obtained in a pure form and specific antibodies could be raised. However, the sample in which such analyte is commonly found usually contains other molecules which are so similar to the analyte that the antibody would cross-react with them. Such a problem is commonly encountered in immunologically measuring isoenzymes. Antibodies specifically raised against one isoenzyme cross-react with many other isoenzymes concurrently present in the sample. Such a problem prevents measurement of the CK-MB isoenzyme by using antibodies raised specifically against it, since such antibodies will cross-react with CK-MM and CK-BB isoenzymes.
A novel method of specifically measuring an analyte which falls into Category c above has been disclosed in a co-pending patent application, Two-Site Cross-reaction Immunometric Sandwich Assay Method, Ser. No. 165,001, filed Jun. 30, 1980. The difference in the prior art and that pending application is that the invention of that patent application requires the use of cross-reactive rather than specific antibodies. The combination of cross-reactive antibodies and adaptation of "sandwich" techniques allows one to specifically measure analytes which herebefore were not specifically measurable by immunological techniques in which specific antibodies were used. Another co-pending patent application. Ser. No. 597,593, Matrix Aided Immunometric Assay for CK-MB, describes the use of a novel matrix to enhance the speed and accuracy of such a test procedure. Yet another application, Two-Site Enzyme Labeled Cross-Reaction Immunometric Sandwich Assay Method, Ser. No. 635,893, filed Jul. 30, 1984 (a CIP of Ser. No. 165,000) describes the use of an enzyme label.
The prior art fails to disclose methods of using cross-reactive antibodies to measure analytes which fall in the Categories a and b above. In the case of Category b, the analyte is reactive and changes from its native analyte form (NAN) to an altered analyte form (abbreviated as AAN) when conjugated for immunization. The antibody thus formed is not specific to the native analyte NAN; however, the antibody would have some cross-reactivity to that molecule. Since such analytes are very reactive, they lend themselves to easy alteration. Thus, a sample containing the analyte of interest often contains both the native (NAN) and altered (AAN) forms of the analyte, and the antibody would react with differing intensities with both forms and would not be suitable for measuring these forms.
Roberts et al. (Lancet, Vol. II, Pages 319-321, August 1977) have described the use of cross-reactive antibodies. They use antibodies raised against CK-BB (creatine kinase, BB isomer) to measure CK-MB (creatine kinase, MB isomer). Antibodies to CK-BB are not raised against CK-MB, they cross-react with CK-MB. The difference between teachings of Roberts and the present invention lies in the fact that two analytes, for example CK-BB and CK-MB, one specific to the antibody and one cross-reactive with the antibody are left unaltered by Roberts and both of them are measured. However, because both analytes do not react similarly, the accuracy of quantitation is affected by the relative proportions of each analyte. In the present invention advantage is taken of the relative instability of the analyte of interest to alter it prior to or during the immunoassay procedure so that it is made specific to the cross-reactive antibody. The cross-reactive antibody is thus made useful instead of being a nuisance and since both forms are converted into one form, quantitation is accurate. This is achieved by creating a matrix in which the native analyte of interest is altered rapidly and thus made specific to the antibody that was formed. The novel feature of this invention is that the antibody is not purified but that the analyte is changed to make it specifically reactive to the cross-reactive antibody.
One specific utility of this novel approach is for the detection of allergy causing antibiotics contamination. The utility of this invention is demonstrated through the detection of penicillin contamination in milk. However, the teaching of this invention is applicable to all beta-lactam antibiotics. The present invention could be used for a relatively simple and inexpensive method for detecting the presence of similar beta-lactam antibiotics in other products.
Products contaminated with beta-lactam antibiotics cause allergenic reactions in some individuals. Also, the presence of such compounds may make it impossible to use such a product for its ultimate use. For example, milk contaminated with penicillin may not be suitable for manufacture of cultured cheese products.
Present methods for detecting antibiotics generally require microbial assays and, hence, considerable time and laboratory facilities are necessary. Present chemical tests do not have sufficient sensitivity to detect the minimum tolerable levels of antibiotic concentration.
It is known that the beta-lactam ring of penicillin and other such antibiotics is very reactive. Thus, the native penicillin may undergo changes, particularly through opening of the beta-lactam ring. Such conversion depends upon the environmental conditions such as pH, temperature, presence of sugars, proteins etc. to which the antibiotic is exposed. Thus, the degree of conversion may be different when it is injected into animals to treat infection, from when it is present in milk or from when it is present in fermentation broth.
While immunological methods for detecting penicillin and similar antibiotics have been described in prior art such as U.S. Pat. No. 4,347,312, or in commercial products such as the SPOT test from Angenics (100 Inman Street, Cambridge MA 02139), they do not teach easy-to-use immunological methods capable of measuring the antibiotic which may be present in both the open beta-lactam ring (e.g. penicilloyl form) and closed beta-lactam ring structure (e.g. native penicillin).
It is well accepted that the open beta-lactam ring form (e.g. penicilloyl group) is the key determinant in antibiotic allergy. Antibodies used to immunologically measure antibiotics (e.g. penicillin) such as that described in U.S. Pat. No. 4,347,312, are usually formed in such a way that they are specific to the open beta-lactam ring (e.g. penicilloyl group) rather than native molecule (e.g. penicillin). Authors such as Kitagawa et al., A New Method for Preparation of an Antiserum to Penicillin and Its Application for Novel Enzyme Immunoassay of Penicillin, Journal of Biochemistry, Volume 84, Page 491, 1978, have recognized this and have developed methods for producing antibodies specific for the native molecule. Jean-Michel Wal et al., Radioimmunoassay of Penicilloyl Groups in Biological Fluids, FEBS Letters, Volume 57 (1), Page 9, 1978, and Anna Moer Isetta et al., Affinity and Specificity of Penicillin-Antibody Interaction Determined by an Enzyme Immunoassay, Eur. Journal of Immunology, Volume 6, Page 737, 1976 on the other hand described methods of producing antibodies against the open beta-lactam ring derivative. Thus, the prior art describes methods for immunologically measuring either the open beta lactam ring (e.g. penicilloyl derivatives) or the closed beta-lactam ring (e.g. penicillin) but not both forms simultaneously.
The opening of the beta-lactam ring causes the antibiotic (e.g. penicillin) molecule to loose its antibacterial activity. Thus, conventional microbiological methods measure native antibiotics (e.g. penicillin) rather than the open beta-lactam ring (e.g. penicilloyl) derivatives. The native antibiotic (e.g. penicillin) still has a very reactive beta-lactam ring and is capable of forming allergenic antigens when it could combine with other molecules to form open beta-lactam derivatives. Thus, microbiological methods for measuring antibiotics contamination give only a partial and an indirect measure of allergenicity of such products.
The allergic reaction in humans, which could be caused by food or drug products contaminated with penicillin or other beta-lactam antibiotics, may result from both native form as well as the altered derivatives. This is because the human body will convert some of the native antibiotic into allergenic derivatives in-vivo. It would be useful therefore to simultaneously measure both the altered form which is allergenic and the intact form which has potential allergenicity. However, the prior art does not describe a method capable of measuring simultaneously both forms of antibiotics, namely those containing an intact beta-lactam ring and those containing open beta-lactam ring derivatives.
The present invention provides means of immunologically measuring both the closed ring antibiotics and the open beta-lactam ring derivative. This is achieved by converting the closed ring antibiotic molecule in the sample into its open beta-lactam ring form. Such a measurement of both forms would provide a more reliable estimate of allergenicity of penicillin and similar beta-lactam ring antibiotics when present as contaminants in foods and drugs.